GSM7088393: WT2-LSC Ribo-seq; Mus musculus; OTHER
3 millions cells were lysed with 300μl lysis buffer (CaCl2 4 mM, MgCl 10 mM, K-HEPES pH 7.2 25 mM, KOAc 200 mM and NP-40 1%) for 5 min on ice and then centrifuged at 9000rpm for 5 min. Collect supernatant and 100 μl of lysate was mixed with TRIzol and saved as input. Adjust KOAc concentration of the lysate to 100 mM by 1:1 dilution with water and then incubated with 10μg/ml micrococcal nuclease for 30 min at 37°C. RNA was extracted with TRIzol. Followed by PNK treatment, ribosome-protected fragments (RPFs), 25 to 40 nucleotides long, was purified with 15% TBE-Urea Gel. Libraries were then prepared using the VAHTSTM Small RNA Library Prep Kit for Illumina® following manufacturer's instructions. Libraries were prepared using the VAHTSTM Small RNA Library Prep Kit for Illumina® following manufacturer's instructions.